The long-term goal of our study is to understand the molecular mechanism of oral carcinogenesis. We have previously investigated the relationship between DNA damage, genetic instability, and development of oral cancer. Among the various types of DNA damage, double-strand breaks (DSBs) are the most common and result in the most deleterious effects. DSBs can be repaired by non-homologous end joining (NHEJ) activities, which may proceed through either error-free or error-prone mechanisms. DSBs repaired by the error-prone NHEJ pathway may lead to accumulation of mutations, resulting in genetic instability of cells. Recently, we developed in vitro and in vivo DNA end-joining assay systems that allowed for distinguishing between error-free and error-prone NHEJ activities. Using these systems, we investigated the differences in the NHEJ activities of normal human oral keratinocytes (NHOK) and human oral squamous cell carcinoma (OSCC) cell lines. The OSCC showed higher error-prone and lower error-free NHEJ activities compared with NHOK. Higher levels of error-prone NHEJ activities were noted primarily in OSCC cell lines with defective p53 compared with those containing wild-type p53. Immortal human oral keratinocytes containing the "high risk" human papillomavirus genome also demonstrated higher levels of error-prone NHEJ activity. These findings suggest the possibility that correctly functioning wild-type p53 may be necessary for error-free NHEJ activities. Accordingly, we hypothesize that OSCC have aberrant DSB repair capacity due to error-prone NHEJ activities, which result from defective p53 function. This hypothesis will be tested in the present proposal with the following specific aims: (1) to investigate error-free and error-prone NHEJ activities, and their relationship with the p53 status in normal and human OSCC cells, (2) to examine whether disrupting p53 affects the NHEJ activities in normal human oral cells expressing wild-type p53, and (3) to identify the p53 domain that is necessary for the error-free NHEJ process. With these specific aims we expect to address the following questions: Are there any differences in the efficiency and fidelity of NHEJ activities in normal and cancer cells? If so, is inactivation of p53 responsible for the differences? Does the disruption of p53 affect NHEJ activities of normal cells? If so, which domain of p53 is necessary for the error free NHEJ process? It is anticipated that the outcome of this proposal will help us advance our research activities regarding the role of p53 in the NHEJ process and genetic stability.